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Analytica Chimica Acta
jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / a c a
Photoactivation by visible light of CdTe quantum dots for inline generation of reactive oxygen species in an automated multipumping flow system
David S.M. Ribeiro, Christian Frigerio, João L.M. Santos, João A.V. Prior
∗Requimte,DepartmentofChemicalSciences,LaboratoryofAppliedChemistry,FacultyofPharmacy,UniversityofPorto,RuadeJorgeViterboFerreirano.228,4050-313Porto,Portugal
h i g h l i g h t s
CdTe quantum dots generate free radicalspeciesuponexposuretovis- ibleradiation.
AhighpowervisibleLEDlampwas usedasphotoirradiationelement.
Thelaboratory-madeLEDphotocat- alyticunitwasimplementedinlinein aMPFS.
Freeradicalspeciesoxidizeluminol producing a strong chemilumines- cenceemission.
Epinephrine scavenges free radical species quenching chemilumines- cenceemission.
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
Articlehistory:
Received1February2012
Receivedinrevisedform17May2012 Accepted18May2012
Available online 27 May 2012
Keywords:
Quantumdots
Visiblelightphotoirradiation Reactiveoxygenspecies Chemiluminescence Multipumpingflowsystem Epinephrine
a b s t r a c t
Quantumdots(QD)aresemiconductornanocrystalsabletogeneratefreeradicalspeciesuponexposure toanelectromagneticradiation,usuallyintheultravioletwavelengthrange.Inthiswork,CdTeQDwere usedashighlyreactiveoxygenspecies(ROS)generatorsforthecontrolofpharmaceuticalformulations containingepinephrine.Thedevelopedapproachwasbasedonthechemiluminometricmonitoringof thequenchingeffectofepinephrineontheoxidationofluminolbytheproducedROS.Duetotherel- ativelylowenergyband-gapofthischalcogenideahighpowervisiblelightemittingdiode(LED)lamp wasusedasphotoirradiationelementandassembledinalaboratory-madephotocatalyticunit.Owingto theveryshortlifetimeofROSandtoensurebothreproduciblegenerationandtime-controlledreaction implementationanddevelopment,allreactionalprocesseswereimplementedinlinebyusinganauto- matedmultipumpingmicro-flowsystem.Alinearworkingrangeforepinephrineconcentrationofupto 2.28×10−6molL−1(r=0.9953;n=5)wasverified.Thedeterminationratewasabout79determinations perhourandthedetectionlimitwasabout8.69×10−8molL−1.Theresultsobtainedintheanalysisof epinephrinepharmaceuticalformulationsbyusingtheproposedmethodologywereingoodagreement withthosefurnishedbythereferenceprocedure,withrelativedeviationslowerthan4.80%.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Epinephrine or adrenaline [1-(3,4-dihydroxyphenyl)-2- (methylamino)ethanol] is an hormone produced by suprarenal glands that belongs tothe catecholamines group and plays an
∗Correspondingauthor.Tel.:+351220428670.
E-mailaddress:joaoavp@ff.up.pt(J.A.V.Prior).
importantroleasneurotransmitter.Thisdrugiswidelyusedinthe treatmentofallergicemergencies,status asthmaticus,bronchial asthma,ventricularbradycardia,cardiacarrest,glaucomaandas styptic[1].Thisdrughasalsobeenreportedasbeingusedindrug abusesituationstoobtainstimulanteffectsthroughintravenous injection[2].Duetoitsrelevanttherapeuticalimportance,several methodologieshavebeendevelopedforepinephrinequantifica- tioninpharmaceuticalformulations,suchas,spectrophotometry [3,4], fluorometry [5,6],electrochemiluminescence [7],capillary 0003-2670/$–seefrontmatter© 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aca.2012.05.034
electrophoresiswithultravioletdetection[8]andelectrochemical [9–16]methods. Additionally, somemethods based on distinct flow analysis techniques were also developed, mostly resort- ing toflow injectionanalysis with spectrophotometric [17,18], spectrofluorimetric[19],amperometric[20]andelectrochemilu- minometric[21]detectionandsequentialinjectionanalysiswith potentiometricdetection[22].
Inthiswork,andforthefirsttime,theadvantageousfeatures resultingfromsimultaneouslyexploitingthehighcatalyticactiv- ityofsemiconductornanoparticlesandtheantioxidantcapacityof epinephrine[23]areputinevidence.
Colloidalsemiconductornanocrystals,oftenreferredtoasquan- tumdots(QD),can bedefined asmonodispersed nanoparticles madeofacoreofsemiconductormaterial,surroundedbyacap- pingorganiclayerorpassivatingmolecule,inadiametertypicallyin therange1–10nm.Thesenanostructuredmaterialscompriseele- mentsfromtheperiodicgroupsIIB–VIB(e.g.CdSe,CdTe,CdS,ZnSe), IIIB–VB(e.g.InP,InAs)orIVB–VIB(e.g.PbSe)[24].QDnanoparticles haveimportantintrinsic properties,suchashighphotostability, highquantum yield, size-tunable, narrow and symmetric band emissionand highabsorptioncoefficientacrossa wide spectral range[25].Thesephotophysicalpropertiesofquantumdotshave madethem attractivematerials indiverse fieldsof application, includingtheirutilizationasfluorescenceprobesandbiomarkersin nanomedicineandtheirexploitationaschemosensorsinanalytical chemistry.
Inrecentyears,severalworkshavebeendevelopedinvolving theuseofQDinfluorescenceorchemiluminescencebasedassays fortheanalyticaldeterminationofseveralcompounds[26–35]and otherchemicalspecies, suchas,heavymetals [36–39].In addi- tion,someotherworkshavetheoreticallyexaminedanotherhighly promisingfeatureofQD,whichistheircapacitytogeneratereactive oxygenspecies(ROS)inaqueoussolution[40–42]uponexposureto anelectromagneticradiation.Nevertheless,fromapracticalpoint ofview,theQDpotentialtogenerateoxidizingspecieswasonly exploitedbySilvestreetal.[43]inaworkthataimedatthedeter- minationofthechemicaloxygendemandofwastewaters.
Inthisworkanovelapproachinvolvingtheuseofavisiblelight irradiationunitbasedonaLEDlampwasdevelopedaimingatthe photoactivationofaqueousCdTeQDtogenerateROS.Theenergy ofthevisibleelectromagneticwasmore thanenoughtotrigger theprocessofradicalsgeneration,thusdispensingmakinguseof ahigherenergyandmoreharmfulUVlampemployedinthepre- viouslymentionedstudies[40–43].Theuseofahigh-powerlamp basedonLEDtechnology,insteadofaUVlowpressuremercury lamp,avoidsthedangerousexpositionoftheanalysttoUVradia- tion,aswellasthecumbersomeexcessiveheatingofthesolutions exposedtotheradiationandalsoreducessignificantlytheenergy consumption(upto90%energysavings).Aimingatimplementing anautomaticcontroloftheQDphotoactivationandalsotoreduce analyticalreagentsconsumption,thenovelanalyticalmethodol- ogywasimplementedinamicro-flowsystemthatexploitedthe multipumpingflowconcept(MPFS).
Themain characteristicsofmultipumping [44],suchas, low reagentsconsumption, straightforward automationand control, highportability, versatilityduetoa modularstructure,allowed inthisworktoevaluatetheuseofavisibleLEDlighttophotoac- tivateinlineQD.TotakeadvantageoftheROSformedinlineby thephotoactivationofQD,itwasimplementeda chemilumino- metricapproachforepinephrinedeterminationinpharmaceutical samplesbyexploitingthedrugantioxidantproperties.
Additionally,thehydrodynamiccharacteristicsofMPFS,arising fromthepulsedflowproducedbytheactuationofthemicro-pumps promotedanefficient,reproducibleandhighsample/reagentinter- mixinginfrontofthedetector,whichisparticularlyimportantdue tothenatureoftheshort-livedspeciesinvolvedinthereactional
scheme,allowingtofurtherimprovetheefficiencyandsensitivity ofthechemiluminescencemeasurementsinwhich lightis usu- allygeneratedbyveryfastreactions.Theproposedmethodology wasbasedonthequenchingeffectofepinephrineontheoxidation ofluminolbytheROSspeciesgeneratedbytheQDnanoparticles irradiation.
2. Experimental 2.1. Apparatus
Thedevelopedflowmanifoldcomprisedfoursolenoidmicro- pumps (model 120SP, Bio-ChemValve Inc., Boonton, NJ, USA), which wereofthefixeddisplacementdiaphragm type,deliver- ing10Lperstroke.Automaticcontrolofthemicro-pumpswas accomplishedwithanIntelPentium®basedmicrocomputerusing softwaredevelopedinMicrosoftVisualBasic6.0®.Thesolenoid devices wereactivatedbya homemadepower drivecontrolled throughcommunicationbythecomputerparallelport.Flowlines madeof0.8mmi.d.PTFEtubing,homemadeend-fittings,connec- torsandacrylicconfluencepointswerealsoused.
Thephoto-excitationunit(LED-PEU)consistedina50cmreac- torcoil madeof PTFE tubing(0.8mm i.d.)placed betweentwo high-powerLEDlamps(Parathom®R5040daylight)emittingwhite lightofhighefficiency.
Thedetectorusedtomonitorthechemiluminescencesignalwas aFP-2020Plusmodel(Jasco,Easton,MD,USA)equippedwitha modularflowcellconsistingofahelical0.8mmi.d.PTFEtubewith aninternalvolumeof100Lthatwaspositionedinfrontofahighly sensitivephotomultiplier. Analyticalsignalswererecordedona stripchartrecorder,modelLinseisL250E.
2.2. Samplesandstandards
Allsolutionswerepreparedwithdoublydeionizedwaterand chemicalsofanalyticalgradewereused.
Forthiswork,threedifferentsamplesforintramuscularinjec- tion were obtained. These pharmaceutical formulations had in theircompositionahighcontentofsodiummetabisulfite,which isaddedasformulationpreservative.Asamplepre-treatmentfor theeliminationofsodiummetabisulfitewasperformedbyapplying aprocedureadaptedfromAmorimetal.[22]withsomeimprove- ments.Briefly,afirstintermediatesamplesolutionwasprepared byadding,ina20mLvolumetricflask,anappropriatevolumeof theinjectabledrugand150L of32%HCl (Panreac®)and then the volume was made up to the mark with deionized water.
Afterwards,this solutionwasbubbledwithnitrogenfor 25min toreleasethedissolvedsulfurdioxideoriginatedbyacidification ofthemedium containingmetabisulfite.A secondintermediate samplesolutionwaspreparedbyappropriatedilutionofthepre- vioussamplesolutionina25mLvolumetricflaskandthepHwas adjustedtoapproximately5.7,withdilutedsodiumhydroxidesolu- tion.Finally, a dilution with water wascarried out in orderto obtainanepinephrinecontentincludedintheanalyticalrangeof themethod.
Takingintoaccountthetechnicaldemandsonsampleprepara- tion,asimilarapproachwasconductedforthestandardsolutions.
A2.28×10−3molL−1epinephrinestocksolutionwaspreparedby dissolving12.5mgofthedrugin25mLofwater.Afirstintermediate epinephrinesolutionwithaconcentrationof6.83×10−5molL−1 waspreparedbyappropriatedilutionofthestocksolutionandby adding187.5Lof32%HClandthenthevolumewasmadeupto 25mLwithwater.Asecondintermediateepinephrinesolutionwith aconcentrationof2.28×10−5molL−1waspreparedbyappropri- atedilutionina50.00mLvolumetricflaskofthefirstintermediate
Fig.1. Multipumpingflow system (MPFS).P1–P4, solenoids micro-pumps;X1
andX2,confluencepoints;LED-PEU,photo-excitationunit;D,chemiluminescence detector;S,sample:epinephrine;C,carrier:H2O;QD,quantumdots:sizeparticle of3.00nmwithaconcentrationof1molL−1inwater;L,luminol:1.5mmolL−1in 0.01molL−1NaOH;W,waste.
epinephrinesolutionandthepHwasadjustedtothepHofdoubly deionizedwater,whichwasapproximately5.7,witha0.2molL−1 sodiumhydroxidesolution.Finally,thevolumewascompletedto themarkwithwater.
The working epinephrine standards (1.14×10−7–2.28× 10−6molL−1)werepreparedbyappropriatedilutionofthesecond intermediate solution by transferring aliquots (0.125–2.50mL) intoa seriesof25.00mLvolumetricflasks andthevolumewas subsequentlymadeuptothemarkwithwater.
A1.0×10−2molL−1 luminolstock solutionwaspreparedby dissolving177.1mgof5-amino-2,3-dihydro-1,4-phthalazinedione (Sigma–Aldrich®) in 100mL of 1.75×10−2molL−1 NaOH solu- tion,which wasusedassolventof luminol.Thisstocksolution was stored under refrigeration. A working luminol solution of 1.5×10−3molL−1wasdailypreparedbydilutionofthestocksolu- tionina100.0mLvolumetricflaskandtheconcentrationofNaOH adjustedto1.0×10−2molL−1.Allluminolsolutionswerealways protectedfromthelight.
2.3. ReagentsandsynthesisofCdTequantumdots
In the synthesis of CdTe QD several reagents were used with the following amount: 1.6×10−3mol of sodium boro- hydride (Sigma–Aldrich®), 0.4×10−3mol of tellurium powder, 200mesh(Sigma–Aldrich®),4.0×10−3molofcadmiumchloride (Sigma–Aldrich®)and1.7×10−3molof3-mercaptopropionicacid (Fluka®).Absoluteethanol(Panreac®)wasalsousedinthesynthe- sisprocess.
ForthesynthesisofCdTeQDcappedwith3-mercaptopropionic acid(MPA)theproceduredevelopedbySilvestreetal.[43]was executed,whichinturnwasbasedintheproceduredescribedby Zouetal.[45].
Fortheassays,asolutioncontaining1.00molL−1ofCdTeQD waspreparedbydissolving25.55mgofthesynthesizedandpuri- fiedCdTeQD,withasizeof3.00nm,in50mLofwater.
2.4. Flowmanifold
ThedevelopedflowsystemexploitingtheMPFSapproachforthe epinephrinedeterminationisdepictedinFig.1.Theanalyticalman- ifoldcomprisedfoursolenoidmicro-pumps(P1–P4)usedforinser- tionandpropulsionofthesolutionsofreagents.Precedingtheana- lyticalcycle,allflowtubingwasfilledwiththecorrespondingsolu- tionbyactuatingthecorrespondingmicro-pump.Theanalytical signalbaselinewasestablishedbyinsertionofH2Othroughactua- tionofP3.
The analytical cycle started with the combined insertion of a pre-set number of sample pulses and CdTe QD solutions in confluence point X1, by the simultaneous actuation of micro- pumpsP1andP2 atafixedpulsetimeof1000ms.Subsequently, the micro-pumps P1 and P2 were switched off and by actuat- ing micro-pump P3 the carrier (H2O) was inserted at a fixed pulse time of 1000ms, corresponding to a pulse frequency of 52min−1,whichfixedtheflowrateduringtransporttodetection at0.52mLmin−1.Theinsertednumberofpulsesofcarriersolu- tionwasenoughtoguaranteethetransportofthereactionzone (pre-mixed sample/QD solution) through the photo-excitation unit (LED-PEU) to the point of confluence X2. The inherent inlinesampledilutionwasstrictlycontrolledandconstantinall determinationsensuring thehighrepeatability oftheanalytical measurements.
Sincethelifetimeoffreeradicalsisextremelyshortandalso consideringthepossibilityofself-quenchingbetweenradicals,the tubelengthfromthelastirradiatedpointofthereactorcoiland theconfluencepointX2wasthestrictlynecessarytoconnectthe reactorcoiltotheconfluencepoint(about3cm).Additionally,the conceptofautomatedanalysis,inwhichallflowparametersare maintainedconstant,ensuredthatthenumberofradicalsreaching confluencepointX2wasalwaysthesamebetweenassaysforthe samesampleanalysis.
Following, by the alternated actuation of the micro-pumps P3 and P4 a pre-set number of plugs of luminolsolution were intercalatedwithplugsofthepre-mixedsample/QDsolutionpre- viouslyirradiatedand,immediatelynext,thereactionzonewas transportedthroughtherepeatedactuationofmicro-pumpP3.To assurethatfromthismomenttheestablishedreactionzonewas rapidlycarriedtothedetectorunit,thepulsetimeoftheactuated micro-pumpswasfixedat125ms,correspondingtoaflowrateof 2.18mLmin−1.Thisflowrateallowedthatthetimeelapsedsince thegenerationofradicalsuntilreachingconfluencepointX2was about1s.
3. Resultsanddiscussion 3.1. Mechanismofreaction
ThepotentialofQDfortheformationofreactiveoxygenspecies (ROS)throughphotoactivationwasonlyrecentlyrecognized,by Ipe et al. [40], where it is stated that the absorption of pho- tonswithenergy higherorequaltothequantumdotbandgap energypromotestheformationofanexciton(electron–holepair).
This means that the semiconductor QD under exposure to an ultravioletorvisibleelectromagneticradiationcanpromote the delocalizationofanelectron(e−)fromthevalenceband(vb)tothe conductionband(cb).Thisformedelectron–holepair(ecb−+hvb+) hasredoxpropertiesthat aredependentfromthevalence band andconductionbandenergiesandprincipallyfromtheflat-bands potentials.ChargetransferbetweenQDandthenearbymolecules willcompetewithradiativeandnon-radiativedecayandenergy transfer.
Inthiswork,thepotentialityofsemiconductorquantumdotsto producefreeradicalsuponelectromagneticirradiationinaqueous solutionwasexploitedbystudyingtheinfluenceofsomeimpor- tantcharacteristicsofthesynthesizedQDonoriginatingROS.The methodologywastested byapplyingit in thedeterminationof epinephrinein pharmaceuticalformulations. Thedetermination wasbasedonthequenchingeffectofepinephrineonthechemilu- minescenceemissionofluminoluponitsoxidationbythereactive oxygenspeciesgeneratedthroughthephotoactivationofaqueous CdTeQDnanoparticles.
Oneoftheinnovationsofthisworkwasthephotoactivation ofQDwithahomemadevisiblelightphotocatalyticunitbasedon LEDs,whichpresentssomeadvantagescomparativelytotheuseof UVlight.
TheuseofwhiteLEDs,thatarecoldlamps,doesnotproduce significantvariationsoftemperaturethatoftenoccurswithincan- descentandgas-dischargelamps.Thisway,therearenovariations oftemperaturethatcouldinfluencetheanalyticaldetermination ororiginatetheformationofairbubbles,whenusingflowsystems.
Also,byusingLEDsemittinginthevisiblewavelengthrangethe photodegradationofepinephrinedoesnotoccur,sincetheradia- tionisinsufficientlyenergeticforcausingmoleculardegradation despitebeingsufficient fortheQDphoto-excitation.Thehome- madephoto-excitationunitwasbuiltwithlampsequippedwith highpowerLEDs,withefficientgenerationofwhitelightandwith- outUVornear-IRradiationinthelightbeam.Theywereofvery lowenergyconsumption(upto90%energysavings)andlong-life, fillingtherequiredstandardsforenvironmentalfriendliness.
The epinephrine determination method was accomplished through3distinctsteps:(i)formationofradicals,(ii)oxidationof luminoland(iii)epinephrinequenching.
In thefirst step, QD wereirradiated withvisible light orig- inating theformation of the exciton (ecb−+hvb+) (Eq.(1)).The excitons undergo redox reactions with oxidizing and oxidable speciespresentintheirradiatedmedium,suchasO2andhydrox- ideion(OH−)respectively.Theconductionbandpotential(ecb−) issufficienttoreduceO2tosuperoxideradicals(Eq.(2))andthe valencebandpotential(hvb+)isenoughtooxidizehydroxideions togeneratehydroxylradicals(Eq.(3)).
1ststep: reactive oxygenspecies (ROS) generationfromphoto- excitationofQD
CdTe+hv→CdTe(ecb−+hvb+) (1)
CdTe(ecb−)+O2→O2•− (2)
CdTe(hvb+)+OH−→ •OH (3)
Inthesecondstep,thefreeradicals•OHandO2•−,generated byphotoactivationofQDinaqueoussolution,oxidizeluminolin alkalinemediumtotheaminophthalateionwhichisproducedin anelectronicallyexcitedstateandemitslight(chemiluminescence signal,CL)ondroppingtothegroundstate(Eq.(4)).Intheabsence ofepinephrine,theobtainedchemiluminescencesignalwasmaxi- mum(blanksignal)sinceallthegeneratedradicalswereavailable fortheoxidationofluminol.
2ndstep:oxidationofluminolbyROSandemissionofthephotons (CLsignal)
luminol+
O2−•OH
OH−
−→3−aminophtlate+h (4)
Inthelaststep,epinephrineactedasafreeradicalscavenger producing adrenochrome (Eq. (5)). The scavenging capacity of epinephrineforfreeradicalsresultsinadecreaseoftheluminol oxidationrateproducingapronouncedinhibitionoftheCLsignal.
TheobtainedCLquenchingwasusedtoquantifytheepinephrine contentsinpharmaceuticalformulations.
3rdstep:ROSscavengingcapacityofepinephrine(quenchingofCL signal)
epinephrine+
O2•−•OH →adrenochrome (5)
Fig.2. NormalizedlightabsorptionspectraofthedifferentsizesofQDusedand emissionspectraofthelightemittedbytheLEDlampsofthephoto-excitationunit.
(---)QDsize=1.87nm;(···)QDsize=3.00nm;(———)QDsize=3.71nm;(—)LED- PEU.
3.2. Characterizationofquantumdots
Taking into account somescientific works [46–48], thesize of QD nanoparticles was expected to have a strong influ- enceonthephotochemical andelectrical properties oftheQD, determining thus its reactivity and the magnitude of the ana- lyticalsignal.Supportedbytheliterature [49],thenanoparticle size was determined by thefirst absorption maximum resort- ing tothe following formula:D=(9.8127×10−7)3−(1.7147× 10−3)2+(1.0064)−194.84inwhich D is the diameterof QD (nm)andisthewavelengthofthemaximumabsorbance.
Themolarconcentrationofthenanocrystalsinsolutioncanbe alsoeasilydeterminedbysimplytakinganabsorptionspectrum ofanaqueousCdTeQDsolutionwithaknownmassconcentration andestablishingtheextinctioncoefficient(ε)usingthefollowing formula[35]:
ε=3450E(D)2.4
whereEisthetransitionenergycorrespondingtothefirstabsorp- tionpeakexpressedineV.ByknowingεandtheabsorbanceofCdTe QDsolution,themolarconcentrationwascalculatedthroughthe applicationoftheLambert–Beer’slaw.
Forthiswork,wereobtainedbysynthesis3differentnanopar- ticle sizes of CdTe QD namely 1.87, 3.00 and 3.71nm, which maximumabsorptionwavelengthswereobservedat485,531and 606nm, respectively. In Fig. 2 is depictedthe normalized light absorptionspectraofthedifferentsizesofQDused,andalso,the emissionspectraofthelightemittedbytheLEDlampsofthephoto- excitationunit.
3.3. OptimizationoftheMPFS
Anoptimizationstudywasperformedinordertoevaluatethe influenceintheanalyticalsignalofthe3differentsizedQDabove mentioned and atsame time differentQD concentrationswere assessed.FortheQDwiththesmallersize(1.87nm)aconcentration rangefrom2.50to10.00molL−1wasevaluated,whilefortheQD withhighersize(3.00and3.71nm)theconcentrationrangestudied wasfrom0.25to1.00molL−1.
The influence in the analytical signal of the different QD sizes and molar concentrations was assessed by establish- ing calibration curves with different epinephrine standards (2.28×10−7–2.28×10−6molL−1). The results were analyzed resortingtoa comparisonbetweentheslopesofthecalibration curves. The results revealed that by increasing the size of the nanoparticlesQDtheproductionofROSalsoincreased,sincefor thenanoparticlesofsmallersizeandforeachtestedconcentration
of QD (2.50–10.00molL−1) the scavenger activity of ROS by epinephrinewasobserved, while forQD of highersize and for thesamerangeofQDconcentrations(2.50–10.00molL−1)itwas notobservedanyscavengingactivity,thatis,therewasnodiffer- encebetweentherecorded analyticalsignal inthepresence or absenceofepinephrine.In this lastsituation,therange of con- centrationstestedforepinephrinewasnotsufficienttoscavenge theelevatedquantityoffreeradicalsformedbyphotoactivation oftheQD.Consideringtheselastresults,theconcentrationsofthe solutionsofQDofsize3.00and3.71nmwere10timesreduced (0.25and 1.00molL−1)andtheassays wererepeated.So, one canconcludethattheuseofQDofhigherdimensionsallowsthe useofsmalleramountsofthenanoparticleswhenaimingforthe photo-generationofROS,makingtheproposedmethodologyenvi- ronmentalfriendly by promotingthereduction in thereagents consumption.Theresultsobtainedintheseassaysrevealedthatthe sensitivityoftheproposedmethodologyincreasedwiththecon- centrationofQDofhighersize(3.00and3.71nm).Amorethorough comparisonoftheresultsobtainedwiththeQDofsize3.00and 3.71nmindicatedthatthehighestsensitivitywasobtainedwith theQDof3.00nmdespitetheQDof3.71nmin sizeoriginatea higherproductionoffreeROS(highestblanksignal).
Theresultsdemonstratedthatahighergenerationoffreeradi- calsdoesnotimplieshighersensitivity,sinceifahighquantityof ROSisgeneratedthenitisnecessarytoincreasetheworkingcon- centrationsofepinephrinetobeabletoobtainamoreaccentuated diminishingoftheanalyticalsignal(CLquenching)andasacon- sequencethedetectionlimitincreases.Ifahigherworkingrange ofepinephrineconcentrationswouldbenecessarythenitcouldbe selectedaQDofhighersize.
Taking into account the final application of the developed methodology in pharmaceutical formulations, the QD of size 3.00nm and with a concentration of 1.00molL−1 in solution wereselected,inordertoobtainabettercompromisebetweenthe parametersreagentsconsumption,sensitivityanddetectionlimit.
Thefollowingassayswereconductedaimingtheevaluationofthe influenceontheanalyticalsignalofsomechemicalandphysical parametersinvolvedinthedeterminationofepinephrine,through theproposedMPFS coupledwitha photocatalyticunitemitting visiblelight,suchasconcentrationofluminolandNaOH,sample volume,flowrateduringtheirradiationofQDandalsotheflow rateduringthetransportofthereactionzonetothedetector.The optimizationoftheparametersunderevaluationwasmadewith theobjectiveofbetteragreementbetweensensitivity,sampleand reagentconsumptionanddeterminationrate.
TheinfluenceofluminolandNaOHconcentrationontheanalyt- icalsignalwasassessedoveraconcentrationrangefrom0.5×10−3 to2.5×10−3molL−1and2.75×10−3to5.0×10−2molL−1,respec- tively.Thestudyoftheinfluenceofluminolconcentrationonthe analyticalsignalwasperformedusinga setof fourepinephrine standardsolutions(2.28×10−7–2.28×10−6molL−1)andfixingthe concentrationofNaOHat0.01molL−1.Foreachconcentrationof luminoltested,calibrationcurveswereestablishedforevaluation ofthesensitivityofthemethodologythroughtheanalysisofthe obtainedslopes. The results demonstrated a more pronounced increaseofsensitivityforconcentrationsofluminolfrom0.5×10−3 uptoapproximately1.5×10−3molL−1,whereasforhighercon- centrationsthesensitivitytendedtowardstabilization.Therefore, for posterior assays the concentration of luminol was fixed at 1.5×10−3molL−1.
Inthesameway,thestudyoftheinfluenceofNaOHconcen- trationontheanalytical signalwasexecutedusingasetoffour epinephrinestandardsolutions(2.28×10−7–2.28×10−6molL−1) andbyusingasolutionofluminol1.5×10−3molL−1.Theresults (Fig.3)revealedthatthesensitivityincreasedmarkedlywiththe concentrationof NaOHup to 1.0×10−2molL−1 and for higher
Fig.3. Influenceofsodiumhydroxideconcentrationinthesensitivityofthemethod- ology.
concentrations the increase was less pronounced. Taking into accounttheobtainedresults,theconcentrationofNaOHselected forfurtherassayswas1.0×10−2molL−1.
Theinfluenceofirradiationtimeontheanalyticalsignalwas alsostudiedbyvaryingtheflowrateduringtheirradiationofthe solutioncontainingQD.Inthemultipumpingflowconcepttheflow rateisdirectlydeterminedbytheinternalvolumeofthemicro- pumps(intheproposedMPFSwas10L)andthepulsesfrequency (oftendefinedbythepulsetime).Theirradiationtimewasvery importantbecauseitdeterminedtheextensionofthegeneration of ROSand therefore, theamplitude ofthe chemiluminescence signaloriginatedbytheoxidationofluminolbytheROS.Thisopti- mizationassayinvolvedtheestablishmentofcalibrationcurvesfor eachpulsetimetestedfrom500msto2500ms(correspondingto flow ratesfrom0.92mLmin−1 to0.23mLmin−1).The optimiza- tionoftheflowrate(pulsetime)duringtheirradiationofQDwas performed takinginto accountthe sensitivity(slope of calibra- tioncurves)andthedeterminationrate.Accordingtotheobtained results,thesensitivityofthemethodologyincreasedapproximately 10%byvaryingthepulsetimefrom500to2000ms(correspond- ingtoadecreaseofflowratefrom0.92upto0.28mLmin−1)and then, tended for stabilization.However,the determination rate decreaseduptoapproximately40%withtheincreaseofthepulse time.Inordertoobtainabettercompromisebetweensensitivity anddeterminationrate,apulsetimeof1000ms(correspondingtoa flowrateof0.52mLmin−1)wasselected.Infact,forthispulsetime thesensitivityincreasedabout5%inamaximumof10%,whilethe determinationratedecreasedapproximately17%inamaximumof 40%.
Thestudyoftheinfluenceoftheflowrateduringthetransportof thereactionzonetothedetector,immediatelyuponphotoactiva- tionofQD,wasarelevantparameteralsoevaluatedbecausetheCL signalisoriginatedbyveryfastreactionsandisatransientsignal.
Theflowrateduringtransporttodetectiondeterminedtheresi- dencetimeofthereactionzoneintheflowsystem,conditioningthe lightintensitymeasured.Indeed,dependingonreactionkinetics, flowratesexcessivelyloworhighcouldresultinaCLemissionout- sidethedetector’sflowcell.Thus,someassayswerecarriedinorder tooptimizetheCLemissionaccordingtotheflowrateduringtrans- porttodetection.Intheseassays,fordifferentpulsetimesof125, 250,400,500and750ms(correspondingtoflowratesof2.18,1.50, 1.09,0.92,0.67mLmin−1),acalibrationcurvewasperformedfor epinephrineconcentrationsupto2.28×10−6molL−1.Theresults (Fig.4)demonstratedafastreactionkineticssincethesensitivity (slopeofcalibrationcurves)markedlyincreasedwiththeflowrate (lowerpulsetimes)upto1.50mLmin−1,approximately.Forhigher flowratesitwasobservedthatthegaininsensitivitywasnotso
Fig.4.Influenceoftheflowrateduringthetransportofthereactionzonetothe detectoronthesensitivityofthemethodology.
Fig.5. Influenceofnumberofpulsesonthesensitivityofthemethodology.
pronounced. Considering that higher flow rates resulted in increasedsensitivityandatthesametime,determinationrate,a flowrateof2.18mLmin−1(correspondingapulsetimeof125ms) wasselectedforfurtheroptimizationassays.
Theinfluenceofsamplevolume,definedbythepumpstroke volume(10L)andnumberofpulsesduringsampleinsertion,was studiedbyvaryingthenumberofsamplepulsesfrom4to12,cor- respondingtosolutionvolumescomprisedbetween40and120L.
Theobtainedresults(Fig.5)showedthatthesensitivitymarkedly increasedwiththenumberofsamplepulsesupto8andforhigher valuesslightlydecreased.
Anotherparameterofgreatimportancewastheflowstrategy usedforsamplezoneandluminolintroductionintheflowsys- temattheconfluencepointX2(Fig.1),sinceitcouldinfluencethe degreeofmixtureofthesolutions,andhence,determinethereac- tiondevelopmentbetweenfree ROSandluminolreagent. Thus, someassays were carried out by exploiting two different flow samplingapproaches,morespecificallymergingzonesandbinary sampling.Theoptimizationmethodusedfortheseassayswasthe sameasabove,aimingatthehighersensitivityoftheproposed methodology.Theslopesofthecalibrationcurvesobtainedforeach
Table1
Compilationoftheoptimizationresultscomprisingthephysicalandchemical parameters.
Parameter Studiedvalues Optimized
values
QDnanoparticlesize(nm) 1.87–3.71 3.00
QDconcentration(molL−1) 0.25–10.0 1.00 Luminolconcentration(mmolL−1) 0.50–2.50 1.50 NaOHconcentration(molL−1) 0.00275–0.05 0.01
Numberofpulses 4–12 8
Samplevolume(L) 40–120 80
Pulsetimeduringirradiation(ms) 500–2500 1000 Flowrateduringirradiation(mLmin−1) 0.92–0.23 0.52 Pulsetimeduringdetection(ms) 125–750 125 Flowrateduringdetection(mLmin−1) 2.18–0.67 2.18
samplingstrategyrevealedasensitivityofapproximately4%higher forbinarysamplingrelativelytothemergingzonesapproach.So, theflow samplingstrategy selectedfor thechemical controlof epinephrineformulationswasbinarysampling.Additionally,the exploitationoftheautomatedanalyticalsystemassuredtheuni- formmixingbetweenthesolutionsattheconfluencepointX2in alldeterminations.
Theresultsobtainedintheoptimizationoftheflowparameters arecompiledinTable1.
3.4. Interferents
Thehighcontentofsodiummetabisulfite(preservative)added tothecompositionofthepharmaceuticalsampleswiththeaim topreventtheoxidationofepinephrineinterferedwiththepro- posedmethodology.Thus,asamplepre-treatmentwasrequired asalreadydescribed inthesubchapter“Samples,standardsand reagents”.Inthisprocedure,thenitrogenbubblingtimewasevalu- atedforaperiodoftimebetween5and30min.Thisstudyshowed thatatimeof25minwasrequiredtoreducetheinterferenceof sodiummetabisulfiteintheanalysisoftheinjectablesamples,indi- catingitssuccessfulremoval.
3.5. Analysisofcommercialpharmaceuticalformulations
Afteroptimizationofthephysicalandchemicalparametersof theflow system,it waspossibletoachieve ananalytical linear response range between 1.14×10−7 and 2.28×10−6molL−1 of epinephrine.Thecalibrationcurvewasrepresentedbytheequa- tionCL=65(±4)×LogC+464(±23)(R=0.9953,n=5),in which CLwasthechemiluminescencesignalquenched,expressedin percentageandCwastheepinephrineconcentrationinmolL−1.
Thedetectionlimit calculatedfromtheequationofthecali- brationcurvewasabout8.69×10−8molL−1.Otherflowanalytical methodologies based on different detection methods, found in theliterature,referhigherdetectionlimits,namely,spectropho- tometric (LOD=8.00×10−6molL−1) [18] and potenciometric (LOD=1.80×10−4molL−1)[22],althoughanelectrochemilumino- metricmethodmentionsaLOD=7.00×10−9molL−1[21].
Table2
Comparisonofanalyticalresultsobtainedinthedeterminationofepinephrineinpharmaceuticalformulationsbytheproposedandthereferencemethod.
Pharmaceuticalsample Declareddosagemg/formulation Amountfound(mg/formulation)a R.D.%b
MPFSmethodology Referencemethod
Pharmaceuticalsample1 0.3mg/0.3mL 0.313±0.005 0.31±0.03 2.53
Pharmaceuticalsample2 1mg/1mL 0.97±0.02 0.95±0.03 2.45
Pharmaceuticalsample3 1mg/1mL 1.04±0.06 0.99±0.09 4.80
aMean±t(0.05)(Student’sttest)×(S/√ n).
b Relativedeviationofthedevelopmentmethodregardingthereferenceprocedure.
Inordertoevaluatetheaccuracyofthedevelopedmethodology, and consequently demonstrate itspotential for routine labora- toryprocedures,thedescribedminiaturizedandautomaticflow systemwasusedforthedetermination ofepinephrinein three formulationsofdifferentpharmaceuticallaboratories,beingallof theminjectables,andtheobtainedresultswerecompared with thosefoundbyconductingareferenceprocedure[50].Theresults, summarizedinTable2,showedagoodagreementbetweenboth methods, with relative deviations between 2.53% and 4.80%. A pairedStudent’st-test[51]confirmedthattherewerenostatis- tical differences (testimated=2.255, ttabulated=4.303) betweenthe results obtained by both procedures, for a confidence level of 95%(n=3).
The evaluation of the precision of the proposed methodol- ogyforepinephrinedeterminationinpharmaceuticalformulations wasperformedthroughtherepeatedanalysisofeachinjectable sample (4 consecutive determinations for each sample). The obtainedresultsrevealedagoodrepeatabilitytakingintoaccount the calculated concentration ranges for a confidence level of 95%(Table2).
The proposed MPFS allowed a determination rate of about 79h−1.
4. Conclusions
CdTe-MPA capped semiconductor colloidal nanocrystals can effectivelyparticipateinredoxphotocatalyticprocesseswithsolu- tionsurroundingspecies.Theextension ofradicalgenerationis noticeablyaffectedbynanoparticlessizebeingmorepronounced forbiggerQD.Therearemanyjustificationsforthissize-radical generation dependence including variations on the surface-to- volumeratioandband-bapenergy,occurrenceofsurfacetraps(less pronouncedonbiggerQD),etc.Furtherresearchisbeingcarriedout toexplaintheseresults.
The main features of MPFS such as high portability, versa- tility, straightforward automation and control combined with the efficiency and simplicity of the LED photo-excitation unit and, additionally, the high sensitivity of chemiluminometric detectionmakesthedevelopedanalyticalmethodologyanattrac- tive tool for easily implementing chemical reaction schemes involving photoactivation of CdTe QD nanoparticles with visi- ble radiation.Theautomaticcontrolof parameters,liketime of irradiationand volume ofsolution containingtheQD nanopar- ticles, among others, that influence the ROS generation by photoactivation of QD, exploited through MPFS coupled to a photocatalytic unit, revealed its usefulness to conduct further scientificstudiesinvolvingQDphotoactivationandthereaction mechanisms.
Also,for thefirsttime, avisible lightmodulebasedonLEDs wasimplementedinanautomaticanalyticalmicro-systemexplor- ingthemultipumpingconcept,forthephotoactivationofaqueous CdTeQDnanoparticleswithouttheinfluenceoftemperaturesince the two LED lampsemployed were cold-lamps. So, the devel- oped LED-PEU can be applied in analysis that involve thermo labile substances or in assays that suffer influence from tem- perature variations. Additionally, being a non-hazard radiation theuseinthis workof visibleradiation tophotoactivateQDis expectedtoinspireresearcherstouseautomaticflowsystemscou- pledwithaLED-PEUunittoconductassayswithQDinvolvingits irradiation.
Acknowledgments
DavidS.M.Ribeirothanksthe“Fundac¸ãoparaaCiênciaeTec- nologia”and FSE(Quadro Comunitário de Apoio) for the Ph.D.
grant (SFRH/BD/42571/2007).Thisworkhasbeensupportedby
“Fundac¸ãoparaaCiênciaeaTecnologia”throughgrantno.PEst- C/EQB/LA0006/2011.
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